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Association of Hydrogen Ion Ejection with Oxidative Phosphorylation by Mouse Liver Mitochondria

From the ¹ From the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the Department of Experimental Biology, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203

1. A method for the determination of photophosphorylation, which involves hydrogen ion change, has been applied to the phosphorylation associated with succinate oxidation by rotenone-treated mouse liver mitochondria. The determination of hydrogen ion uptake as a measure of adenosine diphosphate phosphorylation is considered valid since (a) the hydrogen ion uptake observed following ADP addition was completely inhibited by oligomycin, and (b) the values of H⁺:ADP determined by adding a known amount of ADP and measuring pH change were in good agreement with the theoretical values obtained between pH 6.5 and 8.5.

2. The Sr⁺⁺:ADP coefficient calculated from the quotient of Sr⁺⁺:O and ADP:O was used to calculate an ADP:O corrected for energy leaks. A higher Sr⁺⁺:O ratio was obtained in the presence of oligomycin, and, by application of the Sr⁺⁺:ADP quotient to this ratio, a corrected ADP:O could be obtained, which is considered to represent better the potential for oxidative phosphorylation.

3. The stochiometry of proton ejection associated with Sr⁺⁺ accumulation was also determined, and in phosphate medium the value of H⁺:Sr⁺⁺ was 0.67. In the absence of phosphate, the ratios were markedly dependent on the concentration of Sr⁺⁺ and mitochondria. The optimal ratios were observed at a low Sr⁺⁺ concentration under these conditions, and were approximately 1 with tris(hydroxymethyl)aminomethane chloride medium. With tris(hydroxymethyl)methylglycine buffer, the ratios were as great as 2. Acetate shifted the optimal ratio to an even lower Sr⁺⁺ concentration.

These data were examined on the basis of current proposed models for the translocation of ions, and it was concluded that the intramitochondrial pH plays a primary role in the regulation of respiration and cation uptake.

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